Project Summary/Abstract RNA editing is an evolutionarily conserved form of RNA processing that expands the functionality of the transcriptome. The most prevalent example is adenosine-to-inosine (A-to-I) editing wherein adenosine is chemically modified by enzymes to form inosine. The advent of sequencing-based methods has reignited interest in the RNA editing field, which has revealed a large number of previously unknown, edited sites. For example, a recent study identified close to 20,000 A-to-I sites in the human brain transcriptome, many of which were found on repetitive sequences. More studies have shown clinical correlations between A-to-I edited RNA and the onset of cancer and other diseases such as ALS. Surprisingly, we still know very little about how, why and where they happen due to a lack of tools for studying this important modification. Using innovative imaging techniques, we will visualize and quantify these events using oligonucleotide-based RNA fluorescent in situ hybridization (RNA-FISH) in single cells to reveal the localization and mechanism of RNA editing. Ultimately, we believe that the addition of spatial and temporal information for this processing will increase the understanding of this important biological process and its role in gene expression. We have already validated a FISH-based method that can discriminate adenosines from inosines on glutamate receptor 2, a well-validated, partially edited mRNA for which editing results in an important neuronal phenotype. This proposal outlines the next steps for which we will utilize this new tool to explore the subcellular localization of edited transcripts. In the first aim, we will explore the translocation of edited transcripts in response to cellular stress. In aim two, we will characterize the cell-to-cell variability of A-to-I editing in single-cells. Finally, we will explore the role of hyper-edited Alu sequences in regulating the subcellular localization of the transcripts they are inserted in. Successful completion of these aims will provide an important tool for visualizing A-to-I edited transcripts in single cells, and define changes in subcellular localization of RNA as a function of editing. .